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Background: The non-dairy sector is growing, fermented alternatives to dairy are sparse. Adapted starter cultures to substituting raw materials needs to be developed. Objective: Aims of this study were to isolate, identify, and phenotypically characterize lactic acid bacteria (LAB) that inhabit Swedish legumes, and assess properties necessary for selecting strains with the ability to ferment a bean beverage and with potential health beneficial properties. Design: Isolates of presumed LAB were obtained from legumes collected at Oland, Sweden. Strain diversity was assessed by repetitive polymerase chain reaction (rep-PCR). The strains were identified using matrix- assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS). Species belonging to Enterococcus were predominant along with Pediococcus and closely related Bacillus. Strains were tested for tolerance to low pH, phenol, and bile as well as their bile salt hydrolase (BSH) activity. In addition, Enterococcus strains were tested for antibiotic resistance, and Pediococcus strains for their ability to ferment a bean beverage. Results: From the 25 strains char acterized, five were found resistant to low pH, bile, and phenol, suggesting that they can survive a passage through the gastrointestinal tract (GIT) and hence potentially exert beneficial effects in the host. These are suggested for further investigation on specific host-beneficial properties. Two of these, belonging to Pediococ cus pentosaceus, were able to ferment a bean beverage without any added nutrients, indicating that the Pediococcus strains are well adapted to the bean substrate. One of the P. pentosaceus strains were also able to markedly improve the reduction of phytate by the phytase-producing yeast strain Pichia kudriavzevii TY1322 during co-fermentation as well as increase the final cell count of the yeast strain. Conclusion: Strain isolation and characterization performed in this study aids in selecting starter cultures for legume fermentation. Nutritional properties can be improved by co-fermentation with yeast indicating that novel nutritious fermented non-dairy products could be developed.

Bilberries are a rich natural source of phenolic compounds, especially anthocyanins. The press cake obtained during the processing of bilberry juice is a potential source of phytochemicals. The objective of this study was to evaluate different drying techniques and the fractionation of bilberry press cake powder toward obtaining phenolic-rich ingredients for incorporation into value-added food products. The derived powders were dispersed in water and dairy cream, to investigate the effects of drying and fractionation on the dispersibility and solubility of phenolic compounds. The drying techniques, hot air drying and microwave drying, applied on bilberry press cake reduced the content of total phenolics and anthocyanins. The degradation was, however, consistently small and similar for both techniques. The major anthocyanins detected in the samples were stable during drying and fractionation treatments. Fractionation of the press cake powder affected the total apparent phenolic content and composition of the different fractions. The highest phenolic content (55.33 +/- 0.06 mg g(-1) DW) and highest anthocyanin content (28.15 +/- 0.47 mg g(-1) DW) were found in the fractions with the smallest particle size (

Many studies suggest anthocyanins may prevent the development of several diseases. However, anthocyanin bioactivity against cellular stress is not fully understood. This study aimed to evaluate the protective effect of berry anthocyanins on stressed cells using Saccharomyces cerevisiae. The impact of in vitro gastrointestinal digestion on anthocyanin profiles was also assessed. Bilberry and blackcurrant had higher anthocyanin levels than raspberry and strawberry, but digestion reduced the detected anthocyanins by approximately 90%. Yeast cells with and without digested or nondigested anthocyanin extracts were exposed to H2O2 and examined for survival. In the presence of anthocyanins, particularly from digested strawberry, a significant increase in cell survival was observed, suggesting that the type and levels of anthocyanins are important factors, but they also need to undergo gastrointestinal (GI) structural modifications to induce cell defence. Results also showed that cells need to be exposed to anthocyanins before the stress was applied, suggesting induction of a cellular defence system by anthocyanins or their derivatives rather than by a direct antioxidative effect on H2O2. Overall, data showed that exposure of severely stressed yeast cells to digested berry extracts improved cell survival. The findings also showed the importance of considering gastrointestinal digestion when evaluating anthocyanins’ biological activity.

Berry fruits, such as strawberries and blueberries, are rich sources of anthocyanins. Several studies have been made on the impact of non-thermal treatments on safety, shelf-life and nutritional characteristics of such products, but the effects of these processes on anthocyanin stability during digestion in the gastrointestinal tract are still not completely clear. The aim of this study was to assess the recovery of anthocyanins after simulated gastrointestinal digestion of (1) strawberry samples, pre-treated with pulsed electric field (PEF) at 100 or 200 V&middot, cm&minus, 1, prior to osmotic dehydration (OD), and (2) blueberry samples coated with chitosan and procyanidin. After digestion, a significantly higher content of cyanidin-3-O-glucoside and malvidin-3-O-glucoside was quantified by LC-MS/MS in processed strawberry and blueberry samples, compared with the controls. The highest recovery of cyanidin-3-O-glucoside was detected in digested strawberry samples osmotically dehydrated with trehalose. The recovery of malvidin-3-O-glucoside was highest in digested blueberries coated with chitosan and stored for 14 days, compared with untreated samples or samples coated with chitosan and procyanidin. Our study shows the potential of mild PEF treatments combined with OD, or the use of edible coating, to obtain shelf-stable products without substantially affecting the composition or the stability of anthocyanins during digestion in the upper gastrointestinal tract.

Summary In this work, we present the development and characterization of a strain of Pichia kudriavzevii (TY1322), with highly improved phytate‐degrading capacity. The mutant strain TY1322 shows a biomass‐specific phytate degradation of 1.26 mmol g−1 h−1 after 8 h of cultivation in a high‐phosphate medium, which is about 8 times higher compared with the wild‐type strain. Strain TY1322 was able to grow at low pH (pH 2), at high temperature (46°C) and in the presence of ox bile (2% w/v), indicating this strain's ability to survive passage through the gastrointestinal tract. The purified phytase showed two pH optima, at pH 3.5 and 5.5, and one temperature optimum at 55°C. The lower pH optimum of 3.5 matches the reported pH of the pig stomach, meaning that TY1322 and/or its phytase is highly suitable for use in feed production. Furthermore, P. kudriavzevii TY1322 tolerates ethanol up to 6% (v/v) and shows high osmotic stress tolerance. Owing to the phenotypic characteristics and non‐genetically modified organisms nature of TY1322, this strain show great potential for future uses in (i) cereal fermentations for increased mineral bioavailability, and (ii) feed production to increase the phosphate bioavailability for monogastric animals to reduce the need for artificial phosphate fortification.

Sourdough fermented bread has been suggested to have beneficial health effects, in part mediated by increased satiety in the postprandial phase, but only limited research has been conducted to verify this. The current study aimed to investigate the effect of the amounts of sourdough and rye in soft bread on postprandial appetite. On 6 occasions, 23 healthy volunteers consumed 5 different test breads, with varying amount of rye and sourdough, and a yeast-fermented refined wheat control bread as part of a breakfast meal. The sourdough ranged between 9&ndash, 51% of dough weight and rye content between 35&ndash, 48% of flour weight. Appetite was recorded using visual analogue scales from immediately before breakfast and every 30 min the following 4 h. An ad libitum lunch was served 4 h after the breakfast meal, from which voluntary energy intake was measured. While some of the test breads resulted in lower hunger ratings and increased sense of fullness compared to the refined wheat bread, there were no differences between the test breads. The content of rye in the test breads differed within a narrow range, which might explain the lack of a consistent effect of rye on appetite. Microstructural examination of the test breads showed an increased aggregation of proteins in the breads with high content of sourdough, indicating additional changes to the breads, beyond change in pH, which may counteract the potential effect of decreased pH in the bread on appetite. In conclusion, our study does not support an effect of sourdough on appetite and ad libitum food intake.

Three Gram-stain-positive, non-spore-forming, microaerophilic and fructose-6-phosphate phosphoketolase positive strains were isolated from a faecal sample of an adult subject of the emperor tamarin (Saguinus imperator). Given that the isolates revealed identical BOX PCR profiles, strain TRI 5(T) was selected as a representative and characterized further. Comparative analysis of 16S rRNA gene sequence similarity revealed that strain TRI 5(T) was closely related to Bifidobacterium saguini DSM 23967(T) (96.4 %) and to Bifidobacterium longum subsp. longum ATCC 15708 (96.2 %). Multilocus sequence analyses of five housekeeping genes showed the close phylogenetic relatedness of this strain to Bifidobacterium breve DSM 20213(T) (hsp60 94.1 %), Bifidobacteriumsaguini DSM 23967(T) (clpC 91 %), Bifidobacterium avesanii DSM 100685(T) (dnaG 80.3 %), Bifidobacterium longumsubsp. infantis ATCC 15697(T) (dnaJ 85.3 %) and Bifidobacterium longumsubsp. longum ATCC 15708 (rpoB 93 %), respectively. The peptidoglycan type was A3β, with an interpeptide bridge comprising l-Orn (Lys) - l-Ser - l-Ala - l-Thr - l-Ala. The DNA G+C content of strain TRI 5(T) was 60.9 mol%. Based on the data provided, strain TRI 5(T) represents a novel species of the genus Bifidobacterium for which the name Bifidobacteriumcallitrichidarum sp. nov. is proposed. The type strain is TRI 5(T) (=DSM 103152(T)=JCM 31790(T)).

The influence of supercritical fluid extraction (SFE) and solvent extraction of oils from cloudberry, bilberry, and black currant seeds on the yield, chemical properties, and recovery of antioxidant compounds was investigated. SFE was performed for 1 h at 350 bar and at 50°C and 80°C. Fatty acids, vitamin E, carotenoids, and free radical-scavenging activity (DPPH) were assayed. SFE at 80°C resulted in higher oil yields for cloudberry and black currant seeds. The oils were rich in polyunsaturated fatty acids (PUFAs) (66.8%–75.9% w/w), with high percentages of linoleic and α-linolenic acids. The black currant seed extracts had the highest concentrations of vitamin E (range, 113.0–241.8 mg/100 g oil) and carotenoids (range, 11.5–32.3 mg/100 g oil) and the highest antioxidant activity. The cloudberry seed oils also had high antioxidant content and activity. These findings indicate the potential of SFE for the recovery of PUFA and antioxidant compounds in berry by-products.

Folate is the generic term for a group of chemically related and biologically active forms of vitamin B9, needed by all organisms. Folic acid is the fully oxidized synthetic form of the vitamin. The chapter starts with nomenclature and chemical structure of folate forms followed by methodologies for extraction and chemical analysis of bacterial folate. Folates are unstable and susceptible to interconversion reactions so the special care needed to correctly determine folates in bacteria is discussed. Next follows folate biosynthesis, which happens commonly in plants and many microbes, including most bifidobacteria, but is absent in mammals, such as humans. Relevant genes, enzymes, and intermediate metabolites are described. Thereafter, the chapter continues to review folate metabolism, which describes cycling of different one-carbon units, using folate as vehicle, necessary for, for example, nucleic acid biosynthesis (purines and thymidine), amino acid homeostasis (glycine, serine, and methionine), and methylation of macromolecules, such as proteins and DNA, which includes epigenetic maintenance. Next follows a section on how biotechnology can be applied to produce high levels of microbial folate, to be used either as a food additive or as folate-producing probiotics. Finally, the main concerns and health implications from insufficient folate intake are described.

Bifidobacterial species are widespread in gastrointestinal tracts of mammalian and other animals; they can be found in extra body environment only after a fecal contamination or human intentional addition (as the case of probiotics). Interestingly their occurrence is strictly linked to their hosts with a clear demarcation between animal and human species. PCR-restriction fragment length polymorphism (PCR-RFLP) on the 16S rRNA gene, using Alul, and TaqI restriction enzymes, have been utilized to distinguish the animal or human source of 64 strains belonging to 13 Bifidobacterium species (Delcenserie et al. [15]). Our aim was to test this method updating an in silico restriction analysis on the available 16S rRNA gene sequences of all 55 currently described taxa of Bifidobacterium genus. Our results confirmed the reliability of this method, optimized with the use of three restriction enzymes: Alul, TaqI and MaeIII, as a fast and simple strategy to determine the origin (human or animal) of bifidobacteria. Interestingly, the bifidobacterial species recently isolated from non-human primates cluster in the group of animal source except the bifidobacterial species isolated from higher non-human primates closest to humans such as apes (chimpanzee, orangutan and gorilla) that clusters with human group. Moreover, B. minimum, B. subtile and B. mongoliense isolated only from extrabody environment of which the source is unknown clustered with animal species. The in silico RFLP-PCR confirmed its powerful ability to attribute the primary source of occurrence (human or animal) for bifidobacterial species to the human or animal habitat.

The Yaghnob Valley in Tajikistan is inhabited by a small isolated human population. The area is geographically isolated, causing the inhabitant’s culture, food and lifestyle to remain uninfluenced by the rest of the world. Traditional methods of fermenting milk involve the use of indigenous microorganisms, leading to the production of a variety of fermented milk products. The people in Yaghnobi produce a fermented milk from goat, which constitutes one of their main foods. The aim of the current work was to determine which yeasts were present in the original Yaghnobi fermented milk as well as in the same product reproduced at home for three years with monthly reculturing in cow milk. Thirty yeasts have been isolated using different lab media such as M17, MRS, WL,YPD and YPD plus chloramphenicol, and colonies were re-cultivated until pure. Twenty isolates have been obtained from the original product and 10 from fermented milk reproduced at home. They were identified by Sanger sequencing of the amplified ribosomal Internal Transcribed Spacers (ITS1-4). Furthermore, RFLP analyses of the ITS1-4 region were performed when quality, coverage or similarity of ITS1-4sequences did not permitted the unambiguously identification of the yeast isolates. Only for Saccharomyces cerevisiae, identity was also confirmed with GTG5Rep PCR. The results showed the presence of Kluyveromycesmarxianus (11), Pichia fermentans (7) and Saccharomyces cerevisiae (10) in both products. However, Kluyveromyceslactis (1) was found in the original fermented milk sample and Kazachstaniaunispora(1) in home maintained fermented milk. Great attention has nowadays been devoted to microbial resources and the study of traditional fermented products can be a very interesting source of microbial biodiversity. This study provides for the first time data on yeast composition and characteristics in naturally Yaghnobi fermented milk, showing an unexpected richness in yeast communities leading to fermented milk production.

Background: Bifidobacteria, one of the most common bacteria of the intestinal tract, help establish balance in the gut microbiota and confer health benefits to the host. One beneficial property is folate biosynthesis, which is dependent on species and strains. It is unclear whether the diversity in folate biosynthesis is due to the adaptation of the bifidobacteria to the host diet or whether it is related to the phylogeny of the animal host. To date, folate production has been studied in the bifidobacteria of omnivorous, and a few herbivorous, non-primate hosts and humans, but not in carnivores, non-human primates and insects. In our study we screened folate content and composition in bifidobacteria isolated from carnivores (dog and cheetah), Hominoidea omnivorous non-human primates (chimpanzee and orangutan) and nectarivorous insects (honey bee). Results: Bifidobacterium pseudolongum subsp. globosum, a species typically found in non-primates, was isolated from dog and cheetah, and Bifidobacterium adolescentis and Bifidobacterium dentium, species typically found in humans, were respectively obtained from orangutan and chimpanzee. Evidence of folate biosynthesis was found in bifidobacteria isolated from non-human primates, but not from the bifidobacteria of carnivores and honey-bee. On comparing species from different hosts, such as poultry and herbivorous/omnivorous non-primates, it would appear that folate production is characteristic of primate (human and non-human) bifidobacteria but not of non-primate. Isolates from orangutan and chimpanzee had a high total folate content, the mean values being 7792 mu g/100 g dry matter (DM) for chimpanzee and 8368 mu g/100 g DM for orangutan. The tetrahydrofolate (H(4)folate) and 5-niethyl-tetrahydrofolate (5-CH3-H(4)folate) distribution varied in the bifidobacteria of the different animal species, but remained similar in the strains of the same species: B. dentium CHZ9 contained the least 5-CH3-H(4)folate (3749 mu/100 g DM), while B. adolescentis ORG10 contained the most (8210 mu g/100 g DM). Conclusion: Our data suggest a correlation between phylogenetic lineage and capacity of folate production by bifidobacteria, rather than with dietary type of the host.

The geographically isolated region of the Yaghnob Valley, Tajikistan, has allowed its inhabitants to maintain a unique culture and lifestyle. Their fermented goat milk constitutes one of the staple foods for the Yaghnob population, and is produced by backslopping, i.e., using the previous fermentation batch to inoculate the new one. This study addresses the yeast composition of the fermented milk, assessing genotypic, and phenotypic properties. The 52 isolates included in this study revealed small species diversity, belonging to Kluyveromyces marxianus, Pichia fermentans, Saccharomyces cerevisiae, and one Kazachstania unispora. The K. marxianus strains showed two different genotypes, one of which never described previously. The two genetically different groups also differed significantly in several phenotypic characteristics, such as tolerance toward high temperatures, low pH, and presence of acid. Microsatellite analysis of the S. cerevisiae strains from this study, compared to 350 previously described strains, attributed the Yaghnobi S. cerevisiae to two different ancestry origins, both distinct from the wine and beer strains, and similar to strains isolated from human and insects feces, suggesting a peculiar origin of these strains, and the existence of a gut reservoir for S. cerevisiae. Our work constitutes a foundation for strain selection for future applications as starter cultures in food fermentations. This work is the first ever on yeast diversity from fermented milk of the previously unexplored area of the Yaghnob Valley.

Aims: To screen 19 strains of bifidobacteria for main folate forms composition in synthetic folate-free and complex folate-containing media. Methods and Results: HPLC was used to analyse deconjugated folates extracted from bacterial biomass. Most strains had a total folate content above 4000 mu g per 100 g dry matter (DM). The highest value of 9295 mu g per 100 g DM was found in Bifidobacterium catenulatum ATCC 27539 and the lowest in Bifidobacterium animalis ssp. animalis ATCC 25527 containing 220 mu g per 100 g DM. Ten strains grew in a synthetic folate-free medium (FFM), showing folate autotrophy and suggesting folate auxotrophy of the remaining nine. In the autotrophic strains, a consistently higher folate level was found in FFM as compared to a more complex folate-containing medium, suggesting reduced requirements for folates in the presence of growth factors otherwise requiring folates for synthesis. The contents of total folate, 5-CH3-H(4)folate and H(4)folate were strain dependent. 5-CH3-H(4)folate dominated in most strains. Conclusions: Our results show that bifidobacteria folate content and composition is dynamic, is strain specific and depends on the medium. Suitable selection of the growth conditions can result in high levels of folate per cell unit biomass. Significance and Impact of the Study: This suggests that certain bifidobacteria may contribute to the folate intake, either directly in foods, such as fermented dairy products, or in the intestine as folate-trophic probiotics or part of the natural microbiota.

The fermented cereal-based gruel togwa is used as weaning food for children in Tanzania. Togwa is rich in minerals but these are often not available for uptake in the human intestine due to natural inhibitors, such as phytate (IP(6)). The yeasts Pichia kudriavzevii TY13, Hanseniaspora guilliermondii TY14 and TY20, isolated from Tanzanian togwa, and selected for high phytase activity in complex yeast medium YPD, were now studied regarding their ability to degrade IP(6) in maize-based model togwa. A modified constitutively high-phytase producing Saccharomyces cerevisiae BY80 and commercial Aspergillus ficuum phytase were included for comparison. In addition, a strain of Lactobacillus plantarum was included in the model-togwa set-up. All yeasts in the study grew and reached final cell density 1.5-2 log units higher than the start value. S. cerevisiae BY80 degraded 85% of the IP(6) in 48 h; the same degradation level as with A. ficuum phytase (89%). Of the togwa-isolated yeasts, P. kudriavzevii TY13 and H. guilliermondii TY14 showed strong phytate degradation in the model-togwa; 95% or more of the initial IP(6) was degraded after 48 h. This corresponds to a remaining level of 0.4 and 0.3μmol IP(6)/g dw. Co-inoculation with L. plantarum did not increase IP(6) degradation. Moreover, fermentation with P. kudriavzevii TY13 yielded a successive increase in inorganic phosphate (P(i)), from 0.7 to 5.4 mM, suggesting a phytase production in TY13 which is fairly insensitive to P(i) repression. The study shows that phytate in a model togwa is available for yeast phytase enzymes, and addresses the importance of strain selection for effectively degrading the phytate. Certain yeasts originating from togwa seem to have developed a natural high phytase production, and P. kudriavzevii TY13 and H. guilliermondii TY14 seem particularly well adapted to phytate degradation in togwa, and is our choice for further studies and strain improvement.

The focus of the present investigation was on the Tanzanian fermented food togwa as a source for dietary iron and zinc, and the potential for mineral availability improvements using selected yeasts. To establish the content of target minerals and main inhibitor for intestinal uptake, iron and zinc as well as the mineral chelating phytic acid, (IP(6) or phytate) were determined in naturally fermented togwa. Yeasts were isolated from sorghum, maize and cassava based togwa, and identified by sequencing the D1/D2 region of the LSU rRNA gene. The isolated yeasts were subsequently screened for phytase activity. The total iron content in sorghum, maize and cassava based togwa were 41.5 (+/-7.2), 85.4 (+/-31.9) and 28.6 (+/-3.8) microg/g dw (dry weight) respectively. The zinc content was 12.3 (+/-3.1), 11.0 (+/-1.1) and 6.4 (+/-4.5) microg/g dw in sorghum, maize and cassava based togwa, and the phytate content in the three varieties were 2.6+/-1.2, 4.7+/-0.8 and 0.4+/-0.4 micromol/g dw respectively. The phytate levels in the sorghum and maize based togwa are expected to substantially reduce the availability of iron. The molar ratio phytate to iron for these two varieties were estimated to be 3.5:1 and 3.1:1 respectively. In general, a phytate to iron molar ratio below 1 is needed to increase the availability of iron. Among 26 isolates, 9 different species could be distinguished: Issatchenkia orientalis, Pichia anomala, Pichia norvegensis, Pichia burtonii, Pichia guilliermondii, Kluyveromyces marxianus, Saccharomyces cerevisiae, Hanseniaspora guilliermondii and Candida glabrata. The strains were screened for phytase activity in YPD supplemented with 0.5 mM IP(6). Of 26 screened strains, the phytase activity was most prominent in strains of I. orientalis and H. guilliermondii. The strains and data constitute a basis for further improvements of iron and zinc bioavailability in togwa.

Tissue transglutaminase (tTG) plays an important role in celiac disease pathology as it catalyzes deamidation and cross-linking of specific gluten peptides and converts them into potent epitopes recognized by intestinal T-cells. We investigated whether synthetic peptides with high affinity to gliadin could alter tTG activity on gliadin and whole gluten digest. The immobilized substrates were incubated with synthetic peptides identified by the phage display technique and a control peptide with no affinity to gliadin. Transglutaminase activity was measured with time resolved fluorescence. The mean tTG activity, compared to that of the control without the peptides, was reduced by 31, 33, and 36% for three selected gliadin-binding peptides, and 30% for the peptide pool (Por = 0.001-0.004) when gliadin was the substrate. Finally, substrate specificity experiments suggested that avenin was processed in a manner similar that used for gliadin during in vitro assays with tTG. The results showed that the blocking peptides efficiently reduced tTG processing of gliadin in vitro, and this strategy will be further investigated as an alternative therapy for celiac disease.

Phytases are nutritionally important for increased bioavailability of dietary minerals and phosphate for monogastric animals including humans. Release of minerals and phosphate is accomplished by the enzymatic stepwise degradation of phytate (inositol hexaphosphate, IP6). Activity determinations of phytase is often based on analysis of total released phosphate (Pi), but phytase activity in its purest form represents released product per time from IP6 only. Microbial and plant preparations often also contain mixtures of phosphatases and organic phosphate compounds; hence some released phosphate in enzymatic assays may originate from non-phytase phosphatases degrading non-phytate molecules. Moreover, even purified enzyme extracts assessed via Pi release may result in errors, since commercial IP6 commonly contains contamination of lower inositol phosphates, and further, the products of phytase IP6 hydrolysis are also substrates for the phytase. These facts motivate a quantitative comparative study. We compared enzyme activity determination in phytase assay samples at four different time points, based on analyzing the substrate IP6 versus the product Pi using different selected methods. The calculated activities varied substantially. For example, at 15 min into enzymatic assay, variations from 152 mU/ml (by IP6 analysis on HPIC) to 275-586 mU/ml (by Pi analysis using several methods) was detected. Our work emphasizes the importance of defining the type of activity assessed, showing that phytase activity based on released Pi may yield false positive results and/or overestimations. We propose to differentiate between phytase activity, being the activity by which IP6 is degraded, and total inositol phosphatase activity, corresponding to total released phosphate during the enzymatic reaction.

For the first time to our knowledge, distribution and content of individual folate forms in kefir yeast strains were investigated. This was done using a validated method based on reversed-phase high performance liquid chromatography (HPLC) with fluorescence and diode array (DAD) detection. Eight kefir yeast strains, belonging to different Candida and Saccharomyces species, were isolated from Russian kefir granules. They were grown in synthetic media at standardized conditions before analysis. The average folate content for these yeast strains was 10,780±550 μg/100 g dry matter. In all yeast strains tested, the most abundant folate forms as percentages were 5-methyltetrahydrofolate (43–59%), and 5-formyltetrahydrofolate (23–38%), whereas tetrahydrafolate occurred in a lesser proportion (19–23%).

Lactic acid (LA) has been proposed to be an enhancer for dietary iron absorption, but contradictory results have also been reported. In the present study, fully differentiated Caco-2 cell monolayers were used to evaluate the effects of LA (1-50 mmol/L) on the cellular retention and transepithelial transport of soluble non-heme iron (as ferric nitrilotriacetate). Our data revealed a linear decline in Fe(III) retention with respect to the concentration of LA added. In the presence of 50 mmol/L LA, retention of Fe(111) and Fe(II) decreased 57% and 58%, respectively. In contrast, transfer of Fe(111) across the cell monolayer was doubled, while Fe(II) transfer across the cell monolayer decreased 35%. We conclude that LA reduces cellular retention and transepithelial transport of Fe(l I) by Caco-2 cells in a dose-dependent manner. However, while LA also reduces retention of Fe(111) by Caco-2 cells, the transfer of Fe(III) across cell monolayers is enhanced, possibly due to effects on paracellular transport.

We have studied folate content and composition in 44 yeast strains cultivated in a synthetic medium at standardised conditions. Folates were measured by a validated HPLC method. The total folate content ranged from 4000 to 14,500 mu g/100g dry matter showing a large biodiversity among studied yeasts. The forms found were 5-CH3-H(4)folate and H(4)folate, also varying extensively in relative amounts between different strains. Several strains showed a showed two-fold or higher folate content as compared to the control strain-a commercial strain of Baker's yeast. This indicates that folate content in yeast-fermented foods may be more than doubled merely by choosing a proper strain.

To evaluate lactic acid fermentation as a means to increase the availability of iron in carrot juice, two strains (Lactobacillus pentosus FSC1 and Leuconostoc mesenteroides FSC2), two types of carrot juice and two modes of fermentation were compared. Fermentation improved iron solubility up to 30 fold. The total mineral content and the yield of soluble iron differed between the two types of juice. Addition of pectolytic enzyme and cellulase further improved iron solubility in fermented juice by about 10%. L. pentosus FSC1 yielded the largest improvement in soluble iron, which was not simply a result of a decrease in pH. The concentration of soluble iron in Ln. mesenteroides FSC2 fermentation was linearly related to the major acids produced. Besides, the mineral inhibitor phytate was completely degraded in all the fermentations. Lactic acid fermentation strongly improves iron solubility in carrot juice. The level of improvement was strain specific and related to the produced acids rather than a simple pH effect. Composition of carrot juice and addition of viscosity-reducing enzymes also contributed to this improvement. Our study suggests that carrot juice with high mineral availability may be achieved by fermentation using selected starter cultures, substrate and process.

High levels of heavy metals have been found in the surroundings of the closed uranium (U) mine in Ranstad in southern Sweden. These findings cannot be explained entirely by abiotic processes. It was not until recently that biology was taken into account in the discussion about mobilization of metals at this site. It is well known that bacteria produce short-chain organic acids and element-specific ligands (siderophores) that are able to change pH and enhance chelation, which results in increased mobilization of many trace elements. Other (nonessential) elements, such as thallium (Tl), lanthanides, and actinides, may also be mobilized as a result of such bacterial action. This paper describes the mobilization of U and other elements from U ores by bacteria. Three common siderophore-producing bacterial species, Pseudomonas fluorescens, Shewanella putrefaciens and Pseudomonas stutzeri, were incubated in a chemically defined medium supplemented with tailings material with a content of 0.0013% U by weight, which had been exposed to natural weathering for 30 years. Nonleached U ore (0.61% U by weight) from the same area was incubated with P. fluorescens and S. putrefaciens for comparison. P. fluorescens grown with ore caused a change in pH in the growth medium from 4.7 to 9.3, which was significantly higher than for the other two species, for which pH was about 5.2. P. fluorescens was the only species that thrived and mobilized measurable amounts of U from the two ores, leaching out 0.001–0.005% of the total amount of U from both. The release of U is attributed to the production of pyoverdine chelators, since U could not be detected either in sterile controls or in the experiments with the other two bacteria. P. fluorescens also doubled the chromium (Cr) concentration in solution as compared with the sterile controls, whereas P. stutzeri and S. putrefaciens caused a five to sixfold increase in Cr concentration. Thallium, cobalt (Co), zinc (Zn), manganese (Mn), and vanadium (V) concentrations initially resembled those in the sterile controls, but from day 2 of the experiment, a decrease was observed. The difference in leaching behavior between the bacteria used in this study is likely to have been due to the production of different chelators rather than being an effect of pH, since many metals have low solubility at neutral to alkaline pH. This study using laboratory incubations shows that mobilization of U from ore can occur aerobically at neutral to alkaline conditions, which may be an important process behind the high content of heavy metals in the surroundings of the closed U mine at Ranstad.

The effect of different steeping conditions on phytate, β-glucan and vitamin E in barley during malting was studied by a full factorial experiment with three variables (steeping temperature, barley variety and steeping additions). Addition of lactic acid to the steeping water induced a reduction of phytate during steeping and germination, especially in combination with the high steeping temperature (48 °C). Furthermore, lactic acid and high temperature steeping inhibited β-glucanase development, resulting in a well-preserved β-glucan content after germination. When steeping was conducted without addition of lactic acid, the low steeping temperature (15 °C) promoted development of both phytase and β-glucanase activity during germination. A slightly higher level of tocopherols and tocotrienols was observed in samples steeped at 15 °C than in samples steeped at 48 °C. However, addition of lactic acid reduced the amount for both temperatures. When lactic acid bacteria were added to the steeping water none of the parameters studied differed from samples steeped with water only. The results show the possibility of combining phytate degradation with a preserved β-glucan content during malting and can thus be of interest for development of cereal products with improved nutritional value. Copyright © 2004 Society of Chemical Industry

Summary Phytate (inositol hexa phosphate) hydrolysis can occur during food preparation and production and in the intestine, either by phytase from plants, yeasts or other micro-organisms. This degradation is of nutritional importance, because removal of phosphate groups from the inositol ring results in an increased bioavailability of essential dietary minerals. To substantially improve iron absorption the degradation has to be virtually complete. Food processing techniques increasing the activity of the naturally occurring plant phytases are soaking, malting, hydrothermal treatment and fermentation. An alternative is addition of phytases or micro-organisms producing phytase. Phytate degradation in the stomach and small intestine occurs as a result of activity of dietary phytase of plant or microbial origin. The plant phytase is less stable than fungal phytase in the physiological conditions of the intestine. Biotechnologically produced phytases may be possible for use tomorrow in food processing as well as plant raw material and starter cultures with inserted phytase genes.

Aims Mineral deficiencies cause several health problems in the world, especially for populations consuming cereal-based diets rich in the anti-nutrient phytate. Our aim was to characterize the phytate-degrading capacity of the yeast Pichia kudriavzevii TY13 and its secretion of phytase. Methods and Results The phytase activity in cell-free supernatants from cultures with 100% intact cells was 35–190 mU ml−1 depending on the media. The Km was 0·28 mmol l−1 and the specific phytase activity 0·32 U mg−1 total protein. The phytase activity and secretion of extracellular non-cell-bound phytase was affected by the medium phosphate concentrations. Further, addition of yeast extract had a clearly inducing effect, resulting in over 60% of the cultures total phytase activity as non-cell-bound. Conclusions Our study reveals that it is possible to achieve high extracellular phytase activity from the yeast P. kudriavzevii TY13 by proper composition of the growth medium. Significance and Impact of the Study TY13 could be a promising future starter culture for fermented foods with improved mineral bioavailability. Using strains that secrete phytase to the food matrix may significantly improve the phytate degradation by facilitating the enzyme-to-substrate interaction. The secreted non-cell-bound phytase activities by TY13 could further be advantageous in industrial production of phytase.

Phytases hydrolyze myo-inositol 1,2,3,4,5,6-hexaphosphate (IP(6)), yielding lower inositol phosphates and inorganic orthophosphate. Two commercial strains of baker's yeast (Saccharomyces cerevisiae), Y(1) and Y(2), were able to express phytase activity. This was determined by the capacity to grow in a synthetic medium with IP(6) as the sole phosphorus source. IP(6) hydrolysis was rapid for both strains, and after 24 h, all IP(6) was degraded. Control cultures contained inorganic orthophosphate (P(i)) and no IP(6). Growth rate in IP(6) medium was for both strains essentially identical to growth in P(i) medium, indicating a well-adapted metabolism for utilization of phosphorus from IP(6). There was some difference in growth yield (milligrams of biomass per milligram of glucose) between the two strains: 0.95 (Y(1)) and 1.35 (Y(2)) in IP(6) medium and 1.03 and 1. 35, respectively, in P(i) medium. The phytases were of the 3-phytase type, forming mainly DL-Ins(1,2,4,5,6)P(5), DL-Ins(1,2,5,6)P(4), and DL-Ins(1,2,6)P(3).

Summary A wild-type Saccharomyces cerevisiae , strain CBS 7764, isolated from the intestine of rainbow trout, was analyzed with respect to general growth parameters and global protein expression. Characterization of this strain was of interest since previous data show non-typical S. cerevisiae cell surface properties and because data suggest a probiotic potential of CBS 7764. The heat production rate (dQ/dt), monitored by microcalorimetry, showed that the typical growth phases resulting from diauxic growth on glucose were present in the fish isolate. However, CBS 7764 differentiated from a reference strain by becoming limited in the respiratory phase as demonstrated by a plateau in the dQ/dt signal. The global protein expression, as studied by two-dimensional gel electrophoresis (2D-PAGE), revealed a large degree of resemblance of the fish isolate to the reference strain, however, also clear qualitative and quantitative expression differences were detected; e.g. 14% of the proteins differed in expression level by a factor of at least 2. In addition, the fish isolate expressed 12 unique proteins. The heat shock proteins, which for other organisms have been identified as important in mucosal colonization, were generally expressed to a higher level in CBS 7764.

Microcalorimetry has been demonstrated to be a suitable on-line method for monitoring the lipid production phase of oleaginous yeasts. The choice of lipid extraction method for the oil accumulated by oleaginous yeasts is highly important both for accuracy when quantifying the lipid level and determining the fatty acid composition. The energy content of Rhodotorula glutinis increased from 23.0 kJ/g to 30.6 kJ/g dry biomass during the lipid-accumulating phase and was directly correlated to the analysed level of lipids, when an alkaline hydrolysis extraction method was used. Consequently, bomb-calorimetric measurements of the energy content were shown to be an indirect method of quantifying the lipid content in oleaginous yeasts. The fatty acid composition remained rather constant during the batch growth of Rh. glutinis with approximately 70% unsaturated C18 fatty acids. The high energy content as well as the fatty acid composition of Rh. glutinis makes this yeast a better candidate for use as aquaculture feed compared with the commonly used Saccharomyces cerevisiae.

Five different yeast strains isolated from fish, Saccharomyces cerevisiae HF1 and F2, Sc182 (unidentified strain), Rhodotorula rubra and Rhodotorula glutinis , were used in this study. The cell surface hydrophobicity (CSH) was dependent on growth in all cases. Exponential-phase cells were always hydrophobic while stationary-phase cells became hydrophilic. In contrast, two laboratory strains, Saccharomyces cerevisiae Y41 and Debaryomyces hansenii J26 behaved in the opposite manner as previously reported for S. cerevisiae and Candida albicans . This fact, together with microscopic observations, prompts the suggestion that the cessation of population growth (budding) leads to hydrophilic cell differentiation. Irrespective of the degree of hydrophobicity of the surface examined, exponential-phase cells did adhere to a greater extent than stationary-phase cells. These results suggest that hydrophobicity plays an important role in vivo. Since hydrophobic interactions have been suggested to be the most important forces mediating attachment in the aquatic environment, we do believe that these forces mediate the initial events of fish colonization by yeast strains.

Three strains, Rhodotorula rubra, Rhodotorula glutinis, and Candida zeylanoides, isolated from fish, were tested for the expression of putative tissue colonization factors. All strains were able to bind collagen type I, fibronectin, and laminin to various degrees after growing on various solid and broth media, while the binding to collagen type IV was sparse under all conditions tested. For the three strains tested, a very low cell surface hydrophobicity was shown for growth on various solid and broth media. Mostly, the strains also expressed a negatively charged surface. Extracellular protease activity using different substrates was shown for all three strains. Furthermore, two properties related to iron scavenging, i.e., binding of lactoferrin and production of siderophores, were also tested. For the three strains a capacity to bind lactoferrin as well as a capacity to excrete siderophores were demonstrated. Since these different properties have been correlated to virulence and to the capacity of colonization in other organisms, we address the question of whether the expression of these properties in yeasts could contribute to colonization in fish.Key words: marine yeasts, fish colonization.

At present, there are conflicting data regarding whether or not beta-carotene has a positive effect on iron absorption. This study was undertaken to evaluate possible interactions involved in the uptake of beta-carotene and iron in a human intestinal cell model (Caco-2). The Caco-2 cells were incubated with test solutions containing different amounts of ferrous chloride (10-50 microM) and beta-carotene (0.3-2.5 microM) incorporated in synthetic micelles. In the absence of iron, cellular accumulation of beta-carotene from synthetic micelles was proportional (r (2)=0.97, P

The growing awareness of the relationship between diet and health has led to an increasing demand for food products that support health above and beyond providing basic nutrition. Probiotics are live organisms present in foods, which yield health benefits related to their interactions with the gastrointestinal tract. Phytases are a subgroup of phosphatases that catalyse the desphosphorylation of phytate, which reduces its negative impact on mineral bioavailability, and generates lower inositol phosphates. The aims of this investigation were to (i) study the ability of the probiotic candidate Bifidobacterium pseudocatenulatum to degrade phytate in synthetic medium, to (ii) identify the lower inositol phosphates generated, to (iii) study its survival under conditions mimicking gastrointestinal passage and finally to (iv) assess adhesion of the bacteria to Caco-2 cells. The first steps of InsP(6) degradation by B. pseudocatenulatum phytate-degrading enzyme/s were preferentially initiated at the DL-6-position and 5-position of the myo-inositol ring. It suggests that the main InsP(6) degradation pathway by B. pseudocatenulatum by sequential removal of phosphate groups was D/L-Ins(1,2,3,4,5)P(5) or D/L-Ins(1,2,3,4,6)P(5); D/L-Ins(1,2,3,4)P(4); to finally Ins(1,2,3)P(3) and D/L-Ins(1,2,4)P(3)/D/L-Ins(1,3,4)P(3). This human strain also showed a notable tolerance to bile as well as a selective adhesion capacity (adhesion to control surfaces was zero), to human intestinal Caco-2 cells comparable to the commercial probiotic B. lactis. The phytate-degrading activity constitutes a novel metabolic trait which could contribute to the improvement of mineral absorption in the intestine as a nutritional probiotic feature with potential trophic effect in human gut.

We here demonstrate that folate content in yeast fermented food can be dramatically increased by using a proper (i) yeast strain and (ii) cultivation procedure for the selected strain prior to food fermentation. Folate levels were 3 to 5-fold higher in white wheat bread leavened with a Saccharomyces cerevisiae strain CBS7764, cultured in defined medium and harvested in the respiro-fermentative phase of growth prior to dough preparation (135-139 microg/100 dry matter), compared to white wheat bread leavened with commercial Baker's yeast (27-43 microg/100 g). The commercial Baker's yeast strain had been industrially produced, using a fed-batch process, thereafter compressed and stored in the refrigerator until bakings were initiated. This strategy is an attractive alternative to fortification of bread with synthetically produced folic acid. By using a high folate producing strain cultured a suitable way folate levels obtained were in accordance with folic acid content in fortified cereal products.

Folate content in a Saccharomyces cerevisiae strain was monitored during aerobic batch fermentation in synthetic growth medium, yeast peptone dextrose medium, and a molasses based medium. During growth in the synthetic medium large differences in intracellular folate content was observed at different phases. Specific folate levels, expressed per unit biomass, were highest during respiro-fermentative growth (120 microg/g) and decreased during the respiratory and stationary phases. Thus, the physiological state of the cells clearly affects the folate content. This was confirmed in chemostat cultures where total intracellular folate content increased linearly with increasing growth rate (r(2)=0.998), indicating high growth rate i.e. respiro-fermentative growth to be most favourable to obtain high specific folate content. In complex media however, much lower folate content (15-40 microg/g) was found throughout the batch growth. Only minor growth-phase related differences were detected. This shows the impact of cultivation medium on folate content in yeast. To further investigate which components that influence folate content, batch experiments in synthetic medium with addition of specific components were performed. Adding a raw mixture of peptides and amino acids (peptone) decreased folate levels extensively (90%) whereas adding amino acids one-by-one only had minor effects on the intracellular folate content. Furthermore, supplementing synthetic medium with pABA, folate or nucleotides did not change the intracellular folate content. This work constitutes the first steps towards an optimised process for production of natural folates for fortification purposes, as well as an effort to gain fundamental understanding of folate requirements in yeast in relation to environmental conditions.

Phytases are nutritionally important for increased bioavailability of dietary minerals and phosphate for monogastric animals including humans. Release of minerals and phosphate is accomplished by the enzymatic stepwise degradation of phytate (inositol hexaphosphate, IP6). Activity determinations of phytase is often based on analysis of total released phosphate (Pi), but phytase activity in its purest form represents released product per time from IP6 only. Microbial and plant preparations often also contain mixtures of phosphatases and organic phosphate compounds; hence some released phosphate in enzymatic assays may originate from non-phytase phosphatases degrading non-phytate molecules. Moreover, even purified enzyme extracts assessed via Pi release may result in errors, since commercial IP6 commonly contains contamination of lower inositol phosphates, and further, the products of phytase IP6 hydrolysis are also substrates for the phytase. These facts motivate a quantitative comparative study. We compared enzyme activity determination in phytase assay samples at four different time points, based on analyzing the substrate IP6 versus the product Pi using different selected methods. The calculated activities varied substantially. For example, at 15 min into enzymatic assay, variations from 152 mU/ml (by IP6 analysis on HPIC) to 275-586 mU/ml (by Pi analysis using several methods) was detected. Our work emphasizes the importance of defining the type of activity assessed, showing that phytase activity based on released Pi may yield false positive results and/or overestimations. We propose to differentiate between phytase activity, being the activity by which IP6 is degraded, and total inositol phosphatase activity, corresponding to total released phosphate during the enzymatic reaction.

An in vitro digestion/Caco-2 cell model was applied to explore the impact of lactic acid (LA) fermentation by Lactobacillus pentosus FSC1 and Leuconostoc mesenteroides FSC2 on the Fe bioavailability of carrot juice. The redox state of Fe in fermented carrot juice was also assessed as a crucial factor for absorption. LA fermentation was shown to improve mineral solubility to different extents at simulated physiological conditions: Mn (2-fold); Fe (1.5-1.7-fold); Zn (1.2-fold); Cu (1-fold). Soluble Fe2+ was increased about 16-fold by LA fermentation, and about one third of the Fe2+ remained soluble after in vitro digestion (about 4-5-fold higher than in fresh juice). Data on cell-line studies showed a 4-fold increase in the efficiency of Fe uptake, but not in transepithelial transfer by Caco2 cells, as a result of fermentation. The increases in Fe2+ level and the efficiency of cellular Fe uptake were strain-dependent. To sum up the effect on both Fe solubility and cellular uptake efficiency, the amount of cellularly absorbed Fe from Ln. mesenteroides FSC2-fermented juice was about 20 % higher than that from L. pentosus FSC1-fermented juice (22.7 v. 19.2 microg/l juice per mg protein). To conclude, LA fermentation enhanced Fe absorption by Caco-2 cells from carrot juice because of increases in not only Fe solubility after digestion, but also the efficiency of cellular Fe uptake. The fermentation-improved efficiency of Fe uptake was possibly due to the increased level of soluble Fe2+ rather than a being a strain-specific event.

Hydrolysis of extracellular phytate (InsP(6)) by high-phytase yeast strains and survival of yeast cells were studied at simulated digestive conditions using yeast peptone dextrose growth medium and wheat gruel as model meals. An in vitro digestion method was modified to better correlate with the gastric pH gradient following food intake in vivo. High-phytase yeast gave a strong reduction of InsP(6) (up to 60%) in the early gastric phase, as compared to no degradation by wild-type strains. The degree of InsP(6) degradation during digestion was influenced by the type of yeast strain, cell density, and InsP(6) concentration. Despite high InsP(6) solubility, high resistance against proteolysis by pepsin, and high cell survival, degradation in the late gastric and early intestinal phases was insignificant. Dependency on pH for phytase expression and/or activity seemed thus to be an important limiting factor. Although further studies are needed, our results show the potential of using yeast as a phytase carrier in the gastrointestinal tract.

Myo-inositol hexaphosphate (IP6, phytate) is a potent anti-nutritional compound occurring in many plant-based staple foods, limiting the bioavailability of important nutrients such as iron and zinc. The objective of the present study was to investigate different strategies to achieve high and constitutive extracellular IP6 degradation by Baker's yeast, Saccharomyces cerevisiae. By deleting either of the genes PHO80 and PHO85, encoding negative regulators of the transcription of the repressible acid phosphatases (rAPs), the IP6 degradation became constitutive, and the biomass specific IP6 degradation was increased manyfold. In addition, the genes encoding the transcriptional activator Pho4p and the major rAP Pho5p were overexpressed in both a wild-type and a pho80Δ strain, yielding an additional increase in IP6 degradation. It has previously been proved possible to increase human iron bioavailability by degradation of IP6 using microbial phytase. A high-phytase S. cerevisiae strain, without the use of any heterologous DNA, may be a suitable organism for the production of food-grade phytase and for the direct use in food production.

A simplified HPLC method for rapid determination of folates in yeast with ultraviolet and fluorescence detection without sample purification has been developed. By use of the column Aquasil C(18), specially designed for polar analytes, and gradient elution, it was possible to separate and determine five folate derivatives: tetrahydrofolate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate with fluorescence detection, and 10-formylfolic acid and folic acid with ultraviolet detection. The sample preparation required only a small amount of dry yeast (25-50 mg) and included an extraction of folates by heat treatment and deconjugation of folate polyglutamates to monoglutamates with the use of rat serum conjugase. Validation involved investigation of matrix effects, determination of recovery by standard addition method, repeatability, and stability tests. The dominating folate forms in commercial dry baker's yeast were found to be tetrahydrafolate and 5-methyltetrahydrofolate with a total folate content of 2890 microg/100 g (63.4 nmol/g). The simplicity of the method makes it suitable for folate screening studies of different yeast strains.